It is widely known that the hippocampus is critically involved in acquisition and retrieval of episodic memory and the dentate gyrus is the central information processor for sensory inputs in the hippocampus. The glutamatergic mossy cells of the dentate hilus provide an important recurrent excitatory connection to dentate granule cells and hilar GABAergic interneurons along the longitudinal axis of the hippocampus. While the axonal projections of granule cells and hilar interneurons are restricted within a lamellar plane, mossy cells mediate feedforward inhibition and feedback excitation of distal granule cells along the longitudinal axis and may play a significant role in determining the overall excitability of the dentate. It has been suggested that while mossy cell-driven translamellar inhibition of granule cells is dominant in the normal condition, thereby maintaining sparse coding of granule cells, translamellar disinhibition occurs following the loss of vulnerable mossy cells, which may underlie postinjury hippocampal dysfunction. In fact, hilar mossy cells are known to be the most injury-prone hippocampal neurons. Accordingly, it is presumed that translamellar disinhibition would occur following the loss of mossy cells during injurious stimuli, such as epileptic seizure. However, in vivo contribution of mossy cells to dentate gyrus excitability during either memory formation or exposure to intense stimuli has not been addressed. To understand the role of this mossy cell-mediated dentate gyrus network in vivo, we first generated Cre recombinase transgenic mice by using a BAC DNA fragment carrying 5?-transcriptional regulatory region of murine calcitonin receptor-like receptor (Crlr) gene and Cre cDNA. One of the lines, Cre#4688, showed restricted Cre immunoreactivity (IR) only in the dentate hilar cells at 1-2 months of age, which was co-localized not with GAD65/67-IR but with calretinin-IR, suggesting that the Cre positive cells are mossy cells. X-Gal staining of Cre lines crossed with Rosa26 LacZ reporter mice revealed a functional Cre recombination in dentate mossy cells and CA3c cells, with sparse staining in cerebellar nuclei and preoptic nuclei, suggesting that Cre protein is transiently expressed in CA3c cells and other regions before adolescence. We created hippocampal mossy cell-restricted NMDA receptor (NR) knockout mice based on the critical role of NRs on hippocampus-dependent memory. After crossing with a floxed-NMDA receptor R1 (NR1) strain, significant reduction of NR1 mRNA and NR1-IR was found in the dentate hilus of the mutants at 12 weeks of age, using in situ hybridization and immunohistochemistry. Surprisingly, reduction of NR1 mRNA was not evident in area CA3, cerebellar nuclei, and preoptic nuclei, most likely due to the relative inefficiency of Cre-mediated recombination of the floxed NR1 loci. Therefore, our hippocampal mossy cell-restricted NR knockout line could provide an ideal animal model for the study of mossy cell function. Using these mice, Seiichiro Jinde and others are now developing behavioral research strategies to elucidate in vivo NR function of mossy cells in the acquisition and retrieval of context specificity in an associative memory, such as the ability to distinguish two similar input patterns in a context (pattern separation). We are also generating another mutant line with transgenic expression of Cre recombinase-mediated inducible diphtheria toxin receptor (DTR) under control of the alphaCaMKII promoter, which limits expression to neurons of the forebrain. After crossed with the Cre-expressing mouse strain, Cre#4688, the resulting double-transgenic mutant will express DTR in the surface of Cre-expressing cells only in the forebrain. Since expression of DTR allows binding of the B subunit of DT and subsequent receptor-mediated endocytosis, application of diphtheria toxin (DT) induces the selective ablation of Cre-expressing cells. We plan to evaluate the role of mossy cells in dentate excitability and epileptogenesis using these inducible-mossy cell ablation mice.